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Subject: Veterinary Microbiology × Author: DEEPIKA KUMARI, G × End date: 2019 × Start date: 2019 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    MOLECULAR EPIDEMIOLOGY OF CANINE PARVOVIRUS IN ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) DEEPIKA KUMARI, G; Ramani Pushpa, R.N(MAJOR); Subramanyam, K.V.; Srinivasa Rao, T; Satheesh, K
    Canine parvovirus-2 (CPV-2) is one of the most pathogenic viral etiologic agents causing hemorrhagic enteritis in dogs. CPV is a small, non-enveloped, single-stranded DNA virus of approximately 5 kb genome and belongs to the genus Protoparvovirus, in the family Parvoviridae.The objective of the present work is to detect, isolate and characterize circulating CPV in different locations of Andhra Pradesh from fecal samples of diarrhoeic dogs and its phylogenetic characterization. A total of 342 fecal samples were obtained from diarrhoeic dogs and preliminary screening of the samples was carried out by haemagglutination assay (HA) using 0.8 % swine RBC, out of which 71 were positive with a titre ranging from 1 in 32 to 1 in 512 (20.76%). The results of HA was confirmed by haemagglutination inhibition assay (HI) using hyperimmune serum raised in rabbits. All 342 fecal samples were screened by PCR using CPV-2ab primers and 233 samples produced an amplicon size of 681 bp. Genotyping of CPV was done by employing multiplex PCR using CPV-2ab and CPV-2b primer pairs. Out of 233 positive samples, 216 (92.70%) samples produced an amplicon size of 681 bp characteristic of CPV-2a and 17 samples (7.29%) yielded two specific amplicons of 681 bp and 427 bp and thus categorized as CPV-2b. The fecal samples not reacted with CPV-2ab and CPV-2b primers were further analysed by PCR-RFLP using restriction enzyme MboII for the detection of CPV-2c strain. Only one sample reacted with 555 primers produced an amplicon size of 583 bp but remain undigested with restriction enzyme. The overall prevalence rate of CPV in Andhra Pradesh was found to be 68.42%. Virus isolation studies from the PCR positive fecal samples were carried out using CRFK and MDCK cell lines. Successful isolation was observed in both the cell lines with varied cytopathic effects. Sixteen out of thirty four processed fecal samples in CRFK cell lines could produce a mild CPE after 72h post infection (PI) at fifth passage level which include increased granularity, rounding of cells and degenerative changes. Five out of nine CPV positive samples produced marked CPE like increased granularity, rounding of cells and complete detachment of cells in MDCK cell lines after 72h PI at third passage. The presence of virus at each passage level was confirmed by PCR assay using H primers. Transmission electron microscopic studies revealed spherical virus like particles of approximately 20 nm in diameter. The Partial VP2 gene sequencing of eighteen CPV field isolates along with the vaccine strain was performed in AB13730 DNA Analyzer and with Chromas lite software. The VP2 gene sequences were compared with CPV reference strains available in the GenBank by BLASTn analysis and were found to be highly specific revealing a maximum identity of 99.84% to 100% with CPV-2a and 99.35 with CPV-2b. One sample which only reacted with 555 primer on sequence analysis, exhibited a nucleotide homology of 99.66 % with CPV-2a. Multiple sequence alignment for the 18 CPV field isolates along with the vaccine strain was done using Clustal W 1.8 program and compared with the reference strains of CPV. The Partial VP2 nucleotide sequences identity of the field isolates when compared with the reference strains was 99.99 % to 100% and a similar identity was also observed among the field isolates. The nucleotide sequences for local CPV-2a isolates exhibited 99.99 % of homology with those of CPV-2b isolates. The nucleotide homology levels between the vaccine strain (CPV-2) and the analysed CPV-2a, CPV-2b isolates was 99.98 and 99.97 %, respectively. The antigenic types CPV-2a and CPV-2b differed from the original CPV-2 in atleast four amino acid positions of the VP2 capsid protein. Amino acid sequence analysis was done using MEGA 7.0 and revealed that thirteen out of 18 found to be new CPV-2a, one as a variant of new CPV-2a and four were characterised as new CPV-2b. Based on the typing systems using key amino acid positions, 77.77% were new CPV-2a and 22.22% were new CPV-2b with no CPV-2c or original CPV-2 found. The phylogenetic analysis revealed that the 14 new CPV-2a isolates were having close ancestral relationship with the Indian and Chinese strains of CPV-2a whereas CPV-2b strains formed a separate clade with the Indian CPV-2b strains. Seroprevalence of CPV was done by Indirect ELISA and HI for a total of 542 sera samples. Indirect ELISA recorded 94.65% in vaccinated and 72.50% in unvaccinated dogs whereas HI detected 56.10% in vaccinated and 16.42% in unvaccinated dogs. The variation in the detection of CPV antibodies by Indirect ELISA and HI from vaccinated and unvaccinated dogs was statistically significant (P < 0.05).